Construction of copper-induced gene expression platform in Synechocystis sp pcc6803

Abstract

In Synechocystis sp. PCC6803, gene knock-out is the most straightforward and effective method to reveal the physiological function of a gene. Nevertheless, insertion mutants could not be generated for those genes essential to survival. In order V to elucidate the function of such genes in Synechocystis sp. PCC6803, a copper-induced gene expression platform was constructed using the promoter of petE (PpetE). PpetE from Synechocystis sp. PCC6803 and the beta-galactosidase gene (lacZ) from E. coli GM48 were cloned respectively by doing polymerase chain reaction (PCR), and the PpetE was positioned upstream of lacZ. The PpetE-lacZ construct was integrated into the genome of Synechocystis sp. PCC6803 via homologous recombinations. The expression of beta-galactosidase gene was found to be controllable by adjusting the concentration Of CU2+ in medium. In a range from 6 to 400nmol/L, Cu2+ induced the expression of beta-galactosidase in an S-shaped curve, but when the concentration of cu(2+) in medium was below 6nmol/L or above 400nmol/L, the activity of beta-galactosidase was either too low to be detected or too high to be regulated. This copper-induced gene expression platform can be used in the control of some indispensable genes in Synechocystis sp. PCC6803 : eel Is survived in the presence of Cu2+ so that the physiological effect of a gene Could be observed when Cu2+ is removed